Design, Synthesis, and Biophysical Evaluation of Mechanism-Based Probes for Condensation Domains of Nonribosomal Peptide Synthetases

Ce Shi, Bradley R. Miller, Evan M. Alexander, Andrew M. Gulick, Courtney C. Aldrich

Research output: Contribution to journalArticlepeer-review

Abstract

Nonribosomal peptide synthetases (NRPSs) are remarkable modular enzymes that synthesize peptide natural products. The condensation (C) domain catalyzes the key amide bond-forming reaction, but structural characterization with bound donor and acceptor substrates has proven elusive. We describe the chemoenzymatic synthesis of condensation domain probes C1 and C2 designed to cross-link the donor and acceptor substrates within the condensation domain active site. These pantetheine probes contain nonhydrolyzable ketone and α,α-difluoroketone isosteres of the native thioester linkage. Using the bimodular NRPS responsible for synthesis of the siderophore enterobactin as a model system, probe C2 was shown by surface plasmon resonance (SPR) to stabilize an intermolecular interaction between the peptidyl carrier protein (PCP) and C domains in EntB and EntF, respectively, with a dissociation constant of 1-2 nM, whereas the unmodified holo-EntB showed no interaction with EntF. The described condensation domain chemical probes provide powerful tools to study dynamic multifunctional NRPS systems.

Original languageEnglish (US)
Pages (from-to)1813-1819
Number of pages7
JournalACS Chemical Biology
Volume15
Issue number7
DOIs
StatePublished - Jul 17 2020

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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